Creba Isoform

ABSTRACT

The present invention relates generally to a novel CREBa polypeptide isoform, polynucleotides encoding the polypeptide, expression constructs comprising the polynucleotides, host cell transformed or transfected with the polynucleotides, methods for producing the polypeptide, and methods to identify inhibitors of binding between the CREBa and other polypeptides or polynucleotides.

FIELD OF THE INVENTION

The present invention relates to novel polynucleotides encodingpolypeptides which bind to cAMP regulatory DNA sequences.

BACKGROUND OF THE INVENTION

Extracellular signal transduction leading to specific gene expression isoften carried out by a series of enzymatic reactions which ultimatelymodulate activity of nuclear transcription factors. In one such example,extracellular signaling alters cytoplasmic levels of adenosine3',5'-monophosphate (cAMP) which in turn modulates levels of activecAMP-dependent protein kinase (PKA). Once activated, PKA migrates intothe nucleus and phosphorylates transcription factors which recognize DNAsequences common to genes that are regulated by cAMP signaling pathways.The common DNA sequence which permits cAMP regulation of gene expressionhas been designated the cAMP regulatory element (CRE) and thetranscription factors which recognize and bind to the CRE are known asCRE-binding (CREB) proteins. It has been proposed that CREB proteins areordinarily found in association with CRE DNA sequences and that thephosphorylation state of CREB determines the degree to which the proteinis capable of inducing transcription of the associated gene. Oncephosphorylated, CREB is able to bind a CREB-binding protein (CBP) whichpermits interaction of the complex with transcription factor TFIIB.

It is therefore apparent that regulation of the phosphorylation state ofCREB is central to specific gene expression by cAMP. The phosphorylationstate of CREB, however, is not regulated solely by PKA. On the contrary,the degree of CREB phosphorylation is balanced between the activities ofphophatases as well as kinases other than PKA. Thus, while CREB is amajor participant in coordination of cAMP gene expression, CREB activityis subject to concurrent control by enzymes in other, non-cAMP relatedpathways.

Members in the CREB family of proteins contain conserved regions whichcarry out specific functions related to transcriptional activation. Atthe carboxy terminus, all CREB proteins have a leucine zipper regionwhich permits dimerization of CREB with other CREB proteins or otherheterologous transcription factor subunits. Adjacent the leucine zipperregion, CREB proteins are characterized by a region designated thekinase inducible domain (KID) which is subject to phosphorylation bymultiple kinases other than PKA, including for example, protein kinase C(PKC), casein kinase I (CKI) and casein kinase II (CKII), and possiblycalcium-calmodulin dependent kinases I and II. At the amino terminus,CREB proteins each contain a DNA binding domain rich in basic aminoacids. Despite seemingly subtle differences between proteins within thefamily, reports of variation in gene expression suggest that theproteins have unique physiological roles.

BRIEF SUMMARY OF THE INVENTION

In one respect the present invention provides purified and isolatedpolynucleotides (e.g., DNA sequences, RNA transcripts thereof andanti-sense oligonucleotides thereof) encoding a novel mouse cAMPregulatory element binding, designated mCREBa, polypeptide well aspolypeptide variants (including fragments and deletion, substitution,and addition analogs) thereof which display one or more DNA or proteinbinding activities, one or more specific gene transcription modulationactivities, and/or immunological properties specific to mCREBa. DNAbinding properties include recognition of specific DNA sequences throughwhich mCREBa is able to modulate specific gene expression, while proteinbinding properties include interaction with various regulators of mCREBaactivity, including any of a number of protein kinases, as well asinteractions with specific and non-specific transcription factors.Preferred DNA sequences of the invention include genomic and cDNAsequences as well as wholly or partially chemically synthesized DNAsequences. A presently preferred polynucleotide is set out in SEQ IDNO: 1. Plasmid pBSmb3, comprising the preferred cDNA of the invention,in E. coli strain DH5αF' was deposited on Sep. 18, 1996 with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, and assigned Accession Number 98171. Biological replicas (i.e.,copies of isolated DNA sequences made in vivo or in vitro) of DNAsequences of the invention are contemplated. Also provided areautonomously replicating recombinant expression constructs such asplasmid and viral DNA vectors incorporating mCREBa sequences andespecially vectors wherein DNA encoding mCREBa or a mCREBa variant isoperatively linked to an endogenous or exogenous expression control DNAsequence.

According to another aspect of the invention, host cells, especiallyunicellular host cells such as procaryotic and eucaryotic cells, arestably transformed with DNA sequences of the invention in a mannerallowing the desired polypeptides to be expressed therein. Host cells ofthe invention are conspicuously useful in methods for the large scaleproduction of mCREBa and mCREBa variants wherein the cells are grown ina suitable culture medium and the desired polypeptide products areisolated from the cells or from the medium in which the cells are grown.

Novel mCREBa polypeptides of the invention may be obtained as isolatesfrom natural cell sources, but, along with mCREBa variant products, arepreferably produced by recombinant procedures involving host cells ofthe invention. A presently preferred amino acid sequence for a mCREBapolypeptide is set out in SEQ ID NO: 2. The recombinant products may beobtained in fully or partially phosphorylated or dephosphorylated forms,depending on the cell selected for recombinant expression and/orpost-isolation processing. The mCREBa variants of the invention maycomprise mCREBa fragments which include all or part of one or more ofthe domain regions having a biological or immunological property ofmCREBa including, e.g., the ability to bind to a polypeptide orpolynucleotide binding partner of mCREBa and/or inhibit binding ofmCREBa to a natural binding partner. The mCREBa variants of theinvention may also comprise polypeptide analogs wherein one or more ofthe specified amino acids is deleted or replaced: (1) without loss, andpreferably with enhancement, of one or more biological activities orimmunological characteristics specific for mCREBa; or (2) with specificdisablement of a particular polypeptide/polypeptide orpolypeptide/polynucleotide binding function. Analog polypeptidesincluding additional amino acid (e.g., lysine or cysteine) residues thatfacilitate multimer formation are contemplated. Variant mCREBapolypeptides further include fusion polypeptides wherein all or part ofmCREBa is expressed in conjunction with extraneous polypeptidesequences, including, but not limited to, for example poly-histidinetags, biotinylation tags, β-galactosidase chimera, or chimericpolypeptides including one or more the DNA binding or transactivatingdomains from various transcription factors.

Also comprehended by the present invention are antibody substances(e.g., monoclonal and polyclonal antibodies, antibody fragments, singlechain antibodies, chimeric antibodies, CDR-grafted antibodies and thelike) and other binding proteins (e.g., polypeptides and peptides) whichare specific (i.e., non-reactive with previously identified CREBaisoforms to which mCREBa is structurally related) for mCREBa or mCREBavariants. Antibody substances can be developed using isolated natural orrecombinant mCREBa or mCREBa variants. Binding proteins of the inventionare additionally useful for characterization of DNA or polypeptidebinding site structure(s) (e.g., epitopes and/or sensitivity of bindingproperties to modifications in mCREBa amino acid sequence).

Binding proteins are useful, in turn, in compositions for immunizationas well as for purifying polypeptides of the invention and identifyingcells which express the polypeptides on their surfaces. They are alsomanifestly useful in modulating (i.e., blocking, inhibiting orstimulating) DNA and/or polypeptide binding biological activitiesinvolving mCREBa, especially those mCREBa effector functions involved inspecific and non-specific gene expression. Anti-idiotypic antibodiesspecific for anti-mCREBa antibody substances and uses of suchanti-idiotypic antibody substances in modulating gene expression arealso contemplated. Assays for the detection and quantification of mCREBain body fluids, such as serum or cerebrospinal fluid, may involve, forexample, a single antibody substance or multiple antibody substances ina "sandwich" assay format.

The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor mCREBa makes possible the isolation by DNA/DNA hybridization ofgenomic DNA sequences encoding mCREBa and specifying mCREBa expressioncontrol regulatory sequences such as promoters, operators and the like.DNA/DNA hybridization procedures carried out with DNA sequences of theinvention and under stringent conditions are likewise expected to allowthe isolation of DNAs encoding allelic variants of mCREBa, otherstructurally related proteins sharing one or more of the biologicaland/or immunological properties specific to mCREBa, and proteinshomologous to mCREBa from other species. DNAs of the invention areuseful in DNA/RNA hybridization assays to detect the capacity of cellsto express mCREBa. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of mCREBa by thosecells which ordinarily express the same. As another series of examples,knowledge of the DNA and amino acid sequences of mCREBa makes possiblethe generation by recombinant means of mCREBa variants such as hybridfusion proteins characterized by the presence of mCREBa proteinsequences in association with various extraneous polypeptide sequences.

The DNA of the invention also permits identification of novel genesencoding binding partner polypeptides for mCREBa in any of a number ofwell known techniques, including for example, di-hybrid screening andgel overlay assays. The DNA and amino acid sequence information providedby the present invention also makes possible the systematic analysis ofthe structure and function of mCREBa and identification of thosemolecules with which it will interact. The idiotypes of anti-mCREBamonoclonal antibodies of the invention are representative of suchmolecules and may mimic natural binding proteins (peptides andpolypeptides) through which mCREBa biological activities are modulatedor by which mCREBa modulates intracellular events. Alternately, they mayrepresent new classes of modulators of mCREBa activities. Anti-idiotypicantibodies, in turn, may represent new classes of biologically activemCREBa equivalents. In vitro assays for identifying antibodies or othercompounds that modulate the activity of mCREBa may involve, for example,immobilizing mCREBa or a natural binding polypeptide or polynucleotideto which mCREBa binds, detectably labelling the nonimmobilized bindingpartner, incubating the binding partners together and determining theeffect of a test compound on the amount of label bound wherein areduction in the label bound in the presence of the test compoundcompared to the amount of label bound in the absence of the testcompound indicates that the test agent is an inhibitor of mCREBabinding.

In the alternative, DNA of the invention permits identification ofinhibitors of mCREBa binding to other natural binding partners thoughutilization of a split hybrid assay, wherein mCREBa, or a proteinbinding domain fragment thereof, is expressed in a host cell as a fusionprotein in combination with either a DNA binding or transactivatingdomain of one or more transcription factors. A natural binding partnerof mCREBa is also expressed in the same host cell as a fusion protein incombination with either a DNA binding or transactivating domain of atranscription factor (whichever is not incorporated into the mCREBafusion protein). Expression of the two fusion proteins, and subsequentassociation of the binding partners permits association of the DNAbinding and transactivating domains forming an active transcriptionfactor that leads to expression of a repressor protein. The expressedrepressor protein, in turn, prevents expression of a reporter gene, thussignificantly decreasing survival of the host cell. The thus transformedhost cells can then be contacted with putative inhibitors ofmCREBa/binding partner interaction and actual inhibitors identified asthose which prevent mCREBa binding to its partner protein, thuspreventing expression of the repressor protein which cannot thereforeblock expression of the reporter gene. An inhibitor therefore indirectlyprovides for a positive signal generated by expression and detection ofthe particular reporter gene product. There are at least three differenttypes of libraries used for the identification of small moleculemodulators. These include: (1) chemical libraries, (2) natural productlibraries, and (3) combinatorial libraries comprised of random peptides,oligonucleotides or organic molecules. Chemical libraries consist ofstructural analogs of known compounds or compounds that are identifiedas inhibitory via natural product screening. Natural product librariesare collections of microorganisms, animals, plants, or marine organismswhich are used to create mixtures for screening by: (1) fermentation andextraction of broths from soil, plant or marine microorganisms or (2)extraction of plants or marine organisms. Combinatorial libraries arecomposed of large numbers of peptides, oligonucleotides, or organiccompounds as a mixture. They are relatively easy to prepare bytraditional automated synthesis methods, PCR cloning, or proprietarysynthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, muItiparallelsynthetic collection, recombinatorial, polypeptide libraries.

The DNA sequence information provided by the present invention alsomakes possible the development, by homologous recombination or"knockout" strategies see, e.g., Kapecchi, Science, 244: 1288-1292(1989)!, of rodents or rabbits that fail to express a functional mCREBaprotein or that express a variant mCREBa protein. Such rodents areuseful as models for studying the activities of mCREBa and mCREBamodulators in vivo.

DETAILED SUMMARY OF THE INVENTION

The present invention is illustrated by way of the following examples.Example 1 describes isolation of a partial cDNA encoding a novel proteinby virtue of its interaction with CKIδ. Example 2 provides furthercharacterization of the interaction between the novel protein and CKIδ.Example 3 relates to examination of tissue distribution of the novelmCREBa polypeptide. Example 4 addresses isolation of a full length cDNAencoding mCREBa. Example 5 describes generation of antibodiesimmunospecific for mCREBa. Example 6 relates to recombinant expressionof mCREBa. Example 7 describes identification of the DNA binding sitefor mCREBa. Example 8 relates to generation of a high throughputscreening assay for identification of modulators of mCREBa/DNA bindingactivity. Example 9 addresses use of mCREBa in a split hybrid assay toidentify inhibitors of mCREBa/protein interactions.

EXAMPLE 1 Isolation of cDNA Encoding A Protein That Interacts with CKIδ

In order to isolate cDNAs encoding proteins that interact with CKIδ, atwo-hybrid screen was employed using an expression vector encoding aLexA-CKIδ fusion protein as bait. DNA encoding CKIδ was subcloned intothe BamHI site of pBTM116 Bartel, et al., in Cellular Interactions inDevelopment: a Practical Approach, Hartley (ed.), IRL Press; Oxford, pp.153-179 (1993)! by the following method. The coding region of CKIδHU wasinitially modified to incorporate BamHI restriction sites using PCR withprimers EC 140 (SEQ ID NO: 3) and EC141 (SEQ ID NO: 4) which produced a1.3 kbp DNA fragment.

CGCGGATCCTAATGGAGCTGAGAGTCGGG SEQ ID NO: 3

CGCGGATCCGCTCATCGGTGCACGACAGA SEQ ID NO: 4

The amplification reaction included 300 ng CKIδHU template DNA, 1X PCRbuffer (Perkin Elmer-Cetus), 1.5 mM MgCl₂, 200 uM each dATP, dCTP, dGTP,and dTTP, 10 ng/ml each primer and 1 unit AmpliTaq (Perkin Elmer-Cetus).The reaction was carried out with an initial incubation at 94° C. forfour minutes, followed by thirty cycles of incubation at 94° C. for oneminute, 50° C. for two minutes, and 72° C. for four minutes. Theresulting PCR product was digested with BamHI and ligated into plasmidpAS 1 Durfee, et al., Genes and Development 7:555-569 (1993)! such thatthe encoded protein would be expressed containing an influenzahemagglutinin (HA) epitope tag. The resulting plasmid was transformedinto E. coli by standard procedures and expression of the CKIδ fusionprotein was confirmed by immunoblot using monoclonal antibody 12CA7(Boehringer Mannheim) immunospecific for the HA epitope. The BamHIfragment encoding CKIδ was subsequently subcloned into the BamHI site ofpBTM116 to give plasmid pBTMCKIδ which was transformed into an S.cerevisiae strain L40 (MATa his3Δ200 trp1-901 leu2-3,112 ade2LYS2::(lexAop)₄ HIS3 URA3::(1exAop)₈ -1acZ GAL4) to generate strainCKIδ/L40. CKIδ/L40 was subjected to a large scale transformation with acDNA library made from mouse embryos staged at days 9.5 and 10.5. ThecDNA library was prepared in vector pVP16 according to the method ofHollenberg, et al. Mol. Cell. Biol. 15:3813-3822 (1995)!. Approximately40 million transformants were obtained as determined by survival onmedia lacking leucine and tryptophan.

Eighty-eight million transformants were assayed for protein/proteininteraction by plating on selective media lacking leucine, tryptophan,and histidine. The ability of the yeast to grow in the absence ofhistidine suggested an interaction between CKIδ and a protein encoded bya library sequence. Colonies capable of growth on media lackinghistidine were further screened by standard methods for the ability toexpress β-galactosidase encoded by a gene under transcriptional controlof the LexA operator. One hundred of the yeast colonies that turned bluemost rapidly were grown in liquid media lacking leucine and tryptophanand total yeast DNA was prepared by standard methods. The total yeastDNA was used to transform the E. coli strain C600, which lacks theability to grow on media lacking leucine unless a plasmid expressingleucine is present. Bacteria were plated on agar containingcarbenicillin (an ampicillin derivative) and lacking leucine. Individualcolonies that grew under these conditions were grown up and plasmid DNAprepared.

Since many false positives can occur, positive plasmids were retestedfor interaction with the LexA-CKIδ fusion protein, as well as with aLexA-lamin fusion protein as a negative control. Briefly, the isolatedcDNA library plasmid was cotransformed into L40 with either a plasmidencoding LexA-CKI67 or LexA-lamin, three yeast colonies from eachtransformation are picked and tested in a standard β-galactosidaseblue/white assay. Cells that turned blue with LexA-CKIδ but not withLexA-lamin were grown up, the plasmid DNA isolated, and the sequencedetermined by standard methods. Search of the National Center forBiotechnology Information data base indicated three classes of cDNAswere obtained. Collectively, proteins encoded by the DNAs weredesignated CKIδ-delta-interacting proteins or DIPS.

One class of cDNAs, typified by a clone designated DIP25, contained aportion of a cDNA that was related, but not identical to Drosophilatranscription factor dCREBa. The full length clone was thereforedesignated mCREBa and contains two distinct amino acid motifscharacteristic of CREB proteins. A region rich in basic amino acids,presumably a DNA binding region, is located at the amino terminus whilea leucine zipper domain is found at the carboxyl terminus. A secondclass of DNAs was nearly identical to an EST clone T81950, while thethird class showed no homology to any sequences in the databases.

EXAMPLE 2 Characterization Of The Interaction Between the mCREBa DIP25Clone and CKIδ

In order to further characterize the interaction between CKIδ and themCREBa DIP25 fragment, the mouse clone was expressed in E. coli as a GSTfusion protein as described below.

DIP25-encoding DNA was digested with BamHI and EcoRI and anapproximately 500 bp fragment was isolated and ligated into plasmidpGEX3T (Pharmacia, city state) previously digested with BamHI and EcoRI.The resulting plasmid was transformed into E. coli. Protein expressionwas induced with addition of 0.2 mM IPTG to the media and a GST-DIP25protein of approximately 45 kDa was expressed. The protein was purifiedby adsorption on glutathione agarose (Pharmacia) and used to assess thebinding of DIP25 to CKIδ in vitro as follows.

Recombinant CKIδ was produced as a histidine-tagged fusion protein inE.coli, Ausubel, et al., Current Protocols in Molecular Biology, JohnWiley & Sons (1993) pp. 10.11.8-10.11.22!. Briefly, CKIδ was cloned intothe expression vector pET15b (Novagen). An NdeI restriction site wascreated at the 5' end of CKIδ by site directed inutagenesis and anNdeI/BamHI CKIδ fragment was ligated into pET15b previously digestedwith the same two enzymes. Site directed mutagenesis was carried outwith an oligonucleotide designated KME18 (SEQ ID NO: 10) using theMutagene Phagemid In Vitro Mutagenesis Kit, Version 2 (BioRad) accordingto manufacturer's suggested protocol.

GAATCGGGCCGCCGAGATCTCATATGGAGCTGAGAGTC (SEQ ID NO: 10

The resulting plasmid was transformed into bacterial strain BL21DE3 andthe cells grown to optical density (A₆₀₀) of 0.6. The cells were inducedwith 1 mM IPTG for 18 hours at 25° C., harvested, resuspended in breakbuffer (containing 20 mM NaPO₄, pH 8, 300 mM NaCl, 1 mM PMSF, 1 μMaprotein, 1 μM leupeptin) and lysed in a French Press. The lysate wascentrifuged at 100,000×g for 60 minutes and the supernatant loaded ontoa Ni-NTA-Agarose column (QIAGEN, Germany). The column was washed withbreak buffer and protein eluted using a gradient of 0 to 200 mMimidazole. CKI activity associated with CKIδ eluted between 50 and 80 mMimidazole. The histidine-tagged fusion protein was incubated at 4° C. inbinding buffer (50 mM NaCI, 10 mM Tris pH 7.5, 10% glycerol, 0.1%Triton) with the DIP25-GST fusion protein immobilized on glutathioneagarose beads. After an hour incubation, the beads were washed threetimes in binding buffer and bound protein eluted by boiling in SDSsample buffer containing 2% SDS, 20 mM Tris, pH 6.8, 20% glycerol, and0.001% bromphenol blue. A minor fraction of the DIP25-GST fusion proteinbound the histidine-tagged CKIδ while no CKIδ bound to the negativecontrol, immobilized GST alone. The inefficient binding may reflecteither improper folding of the GST fusion protein or it may indicate aneed for some post-translational modification of DIP25 that E. coli areunable to carry out.

EXAMPLE 3 Tissue Distribution of mCREBa

In order to determine the tissue expression pattern of mCREBa mRNA,Northern blot analysis was carried out using containing a commercialmembrane containing poly A⁺ RNA isolated from various mouse tissues andfrom whole mouse embryos at days 7, 11, 15, and 17 in gestation(Clontech, Palo Alto, Calif.). The membrane was probed following themanufacturer's suggested protocol using DNA generated by PCR from themCREBa clone using the primers mCR-1 (SEQ ID NO: 5) and mCR-2 (SEQ IDNO: 6).

GGAATTCGCTCAAGGAGAGTCCTATTGG (SEQ ID NO: 6)

CGGGATCCTCACAGCTCCACATAAGCTGC (SEQ ID NO: 5)

The PCR included 50 ng DIP25 DNA as template, 1× PCR buffer(Perkin-Elmer Cetus), 1.5 mM MgCI₂, 200 μM dATP, 200 μM dGTP, 200 μMdTTP, 1 μM dCTP, 50 μCi α³² P-dCTP, 10 ng/ml each primer, and 1 unitAmpliTaq (Perkin-Elmer Cetus). The reaction was carried out in aPerkin-Elmer Cetus Thermocycler Model 480 as follows: an initialdenaturation cycle at 94° C. for four minutes, followed by 20 cycles of94° C. for 15 seconds, 60° C. for 15 seconds, and 72° C. for 30 seconds.Unincorporated nucleotides were removed using a Nuc-trap Push Column(Stratagene).

Two mRNAs were detected with one migrating at about 8 kb and the otherat about 3.75 kb. The two mRNAs always detected together suggesting twosplice variants. On the tissue RNA membrane, the highest levels ofexpression were seen in kidney, lung, and heart, with lower levelsdetected in skeletal muscle, liver, brain and testis. Very lowexpression was detected in spleen RNA on this particular blot. In themouse embryo samples, expression of both the 8 kb and the 3.75 kb mRNAswas highest at day 7 and lowest at day 11. After day 11, a progressiveincrease in expression was detected up to days 15 and 17.

In situ hybridization of mouse embryos was performed to determine whichembryonic tissues expressed the mCREBa mRNA. Normal Balb/c mouse embryoswere harvested and embedded in optimal temperature compound (TissueTech, Elkhart, Ind.) and whole embryos were sectioned at 6 micronthickness. Tissue sections were placed on Superfrost Plus® (VWRScientific, Seattle, Wash.) and allowed to air dry overnight at roomtemperature. Sections not used immediately thereafter were stored at-70° C. After drying, sections were fixed in 4% paraformaldehyde (Sigma)in PBS for 20 minutes at room temperature, dehydrated using ethanol inincreasing concentrations (70%, 95% and finally 100%) for one minute at4° C. at each concentration, and air dried at room temperature. Thesections were denatured for two minutes at 70° C. in a solutioncontaining 70% formamide in 2× SSC. Sections were rinsed in 2× SSC at 4°C. and dehydrated and air dried again as previously described.Hybridization was carried out using ³⁵ S-labeled single stranded mRNAgenerated from murine DIP25 DNA by in vitro transcription using ³⁵S-dUTP (Amersham). The labeled probe and diethylpyrocarbonate(DEPC)-treated water were added to a hybridization buffer of 50%formamide, 0.3M NaCl, 20 mM Tris, pH 7.5, 10% dextran sulfate, 1×Denhardt's, 100 mM dithiothreitol (DTT) and 5 mM EDTA. Prior toaddition, the probe solution was heated for three minutes at 95° C. todenature the probe, and 20 μl of the buffer was applied to each tissuesection which was then covered with a sterile RNAse free coverslip.Hybridization was carried out overnight at 50° C.

After hybridization, the sections were washed for one hour at roomtemperature in 4× SSC with 10 mM DTT, followed by additional washes for40 minutes at 60° C. in a buffer containing 50% deionized formamide, 2×SSC, and 10 mM DTT and 30 minutes at room temperature in a 0.1× SSCbuffer. The sections were dehydrated and air dried as described aboveand dipped in Kodak (Rochester, N.Y.) NTB2 nuclear emulsion diluted 1:1with 0.6M ammonium acetate at 45° C. Slides were air dried 1 to 2 hoursin the dark and exposed at 4° C. in the dark in the presence of adesiccant. After ten days exposure, the slides were developed in KodakDektol developer, washed with deionized water and submerged in Kodakfixer for four minutes at room temperature. The sections were thencounterstained with hematoxylin and eosin (Sigma, St. Louis, Mo.).Results from this analysis are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Expression of mCREBa mRNA in mouse embryos                                              Day 15                                                                              Day 16     Day 17  Day 18                                     ______________________________________                                        spinal roots                                                                              +       +          +     +                                        peripheral nerves                                                                         +       +          +     +                                        hindbrain   +                                                                 nasal areas +       +                                                         optic areas +                  +     +                                        unidifentified region                                                                             +          +     +                                        of the brain                                                                  tongue              +                                                         bladder                        +     +                                        kidney              +                                                         lung                +                                                         ______________________________________                                    

EXAMPLE 4 Isolation of a cDNA Encoding Full Length mCREBa

In order to obtain a full length cDNA encoding mCREBa, 5.4×10⁶ clonesfrom a mouse brain UniZAP XR cDNA library (Stratagene) were screened byhybridization with a probe generated by PCR from the mCREBa DIP25 cloneusing oligos mCR-1 and mCR-2 as described above. Hybridization wascarried under conditions wherein nitrocellulose filters were incubatedfor 18 hours at 65° C. in 3× SSC, 5× Denhardt's, 0.1% sarkosyl, 20 mMNaPO₄, pH 6.8, 100 μg/ml single stranded salmon sperm DNA. One clonehybridized to the probe. The cDNA, designated pBSmb3, was isolated by invivo excision according to the manufacturer's suggested protocol andsubjected to microsequencing. Sequencing analysis of the cDNA revealed a3190 bp clone containing an open reading frame beginning at nucleotide304 and ending at nucleotide 1866 with a predicted molecular weight ofapproximately 57 kDa. The nucleotide and amino acid sequences of theclone are set forth in SEQ ID NOs: 1 and 2, respectively. The DIP25clone corresponds to nucleotides 1010 through 1514 of the full lengthmCREBa clone pBSmb3, encoding amino acids 237 through 405 representingboth the basic DNA binding domain and the leucine zipper domain of theprotein. Comparison of amino acids 406-508 of the leucine zipper regionin dCREBa to amino acids 260 to 361 in the corresponding region ofmCREBa reveals 78% identity and comparison of these regions atnucleotide level indicates 68% homology between nucleotides 2202 to 2511of dCREBa and nucleotides 1081 to 1386 of mCREBa. In vitro transcriptionand translation of the clone using TNT kit (Promega, Madison, Wis.)produced a protein product that migrated slightly slower than thepredicted molecular weight (about 75 kDa), possibly caused by the highlycharged basic DNA binding domain or the secondary structure formed inthe leucine zipper domain. In addition, a subclone of pBSmb3 wasgenerated by digesting the plasmid with EcoRI and XhoI to produce a 1.8kb fragment that was ligateded into pBluescript sk⁻ previously digestedwith the same two enzymes. The resulting plasmid was designatedpBSmb3E/X. In vitro transcription and translation of the encoded generesulting in production of a polypeptide of approximately 75 kDsuggesting that the entire coding sequences for the protein wascontained on the 1.8 kb fragment. Further, amino acid sequence analysissuggests that, similar to other CREB isoforms, mCREBa may potentially bephosphorylated by kinases other than CKIδ, including but not limited toother CKI isoforms, CKII, cdc2, MAP kinase, and S6 kinase.

EXAMPLE 5 Production and Characterization of mCREBa Antibodies

Polyclonal antibodies specific for mCREBa were generated using theDIP25-GST expression product of plasmid pGEX3T-DIP25 described above.Briefly, C600 bacteria transformed with the plasmid were grown to anabsorbance at 600 nm of 1.8, at which point expression of DIP25-GST wasinduced for five hours by addition of 0.1 mM IPTG which acted on theendogenous tac promoter. Following induction, bacteria were harvested bycentrifugation and resuspended in phosphate buffered saline (PBS)containing 1 mM phenylmethylsulfonylfluoride (PMSF), 1 μg/ml leupeptide,and 1 μg/ml pepstatin. The cells were lysed in a French press andcentrifuged for twenty minutes at 12,000×g. The supernatant wasincubated for one hour with 3 ml of a 50% slurry ofglutathione-Sepharose 4B (Pharmacia), after which the resin was pelletedand washed three times in PBS. Protein was eluted from the resin using50 mM glutathione in 10 mM Tris, pH 8.0.

Female New Zealand White rabbits were initially immunized with 200 μgDIP25-GST antigen mixed with Freund's complete adjuvant injectedsubcutaneously at multiple sites. Subsequent boosters were carried outwith protein that was first boiled in SDS sample buffer, loaded onto anSDS polyacrylamide gel, and electroeluted from gel slices. Boosterantigen preparation was mixed with Fruend's incomplete adjuvant andadministered at approximately 21 day intervals following the firstimmunization. Test bleeds taken after immunizations 3, 4, and 5.

The immunization resulted in two polyclonal antisera designated 6179 and6144. In order to determine if the antisera recognized mCREBa, pBSmb3was transcribed and translated in vitro in the presence of ³⁵S-methionine using a TNT kit (Promega). The translation extract wasdiluted to 0.5 ml with NP40 IBP (containing 1% Nonidet P40, 50 mM Tris,pH 7.5, 100 mM NaCl, 1 mM EDTA) and incubated on ice for one hour with 5μl rabbit antisera. Following incubation, 20 μl of a 50% slurry ofprotein A agarose (Repligen) was added and incubation continued on icefor an additional 20 minutes. Immune complexes were collected bycentrifugation and washed three times in NP40 IPB. Protein was elutedfrom the complexes by boiling in SDS sample buffer and loaded onto anSDS polyacrylamide gel. After separation, the gel was fixed in aceticacid and methanol, treated with the fluor Amplify (Amersham), dried, andexposed to x-ray film. Autoradiography indicated that both antiserareacted with the 79 kDa expression product from pBSmb3E/X describedabove.

In order to determine whether the antisera reacted with mCREB expressedin mammalian cells, an expression plasmid encoding a mammalian CREB wasconstructed as follows. Parental plasmid pcDNA 3 (Invitrogen) wasdigested with restriction enzymes EcoRI and XhoI and ligated with the1.8 kbp EcoRI/XhoI fragment from pBSmb3E/X described above and encodingthe complete mCREB protein. The resulting plasmid was used totransiently transform a human embryonic kidney cell line 293T by themethod of Chen and Okayama Biotechniques 6:632-638 (1988)!. Cell lysateswere generated 48 hours after transformation in buffer containing 1%Triton X-100, 10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM Na₄ P₂O₇, 50 mM NaF, 100 μM Na₃ VO₄, 1 mM PMSF, 1 μg/ml aprotein, and 1 μg/mlleupeptin and the lysate centrifuged at 10,000×g. One hundred μg clearedlysate was loaded onto a 10% SDS polyacrylamide gel and immunoblots wereproduced by standard techniques. Both antisera, diluted 1:1000, reactedwith a protein of approximately 79 kDa protein in the in lysate fromcells transfected with pcDNA3 containing the mCREB sequences.

EXAMPLE 6 Expression of Recombinant mCREBa

Recombinant mCREBa was expressed in E. coli as a fusion with a proteinusing a modified Pinpoint expression vector (Promega) which permitsexpression of a biotinylated protein when the host cells are grown inthe presence of biotin. Briefly, a 1.8 kb NcoI/XhoI fragment was excisedfrom pBSmb3 and subcloned into expression plasmid araBC previouslydigested with NcoI and XhoI. The resulting intermediate plasmidcontained mCREBa-encoding sequences under the control of the arabinosepromoter. In order to fuse a biotin tag to mCREB, an EcoRI/NcoI promoterfragment from the expression plasmid arabio1b was inserted into theintermediate plasmid such that the mCREBa coding sequence was in framewith a biotin tag and under the control of the arabinose promoter. Theresulting plasmid was designated arabiomCREB. Bacteria were transformedwith arabiomCREB by standard techniques and grown to mid-log phase inLuria Broth containing 2 μM biotin. Expression of biotin-mCREBa wasinduced with 1% arabinose, the bacteria harvested by centrifugation, andthe pellet was resuspended in 50 mM Tris. pH 8.0, 100 mM NaCl, 1 mMEDTA, 5 mM EGTA, and 1 mM DTT. The cells were lysed in a French Press,insoluble material removed by centrifugation, and the supernatant wasadjusted to 150 mM NaCl, 5% glycerol, 0.1% Tween 20, 4 mM DTT. Thebiotinylated mCREBa was purified by addition of streptavidin-agarose(Promega) and incubation for 4 hours at 4° C. with rocking. Boundprotein was washed three times with buffer containing 50 mM Tris, pH8.0,100 mM NaCl, 1 mM EDTA, 5 mM EGTA, and 1 mM DTT. Glycerol was added to afinal concentration of 10% and the biotin-mCREBa protein bound tostreptavidin-agarose stored at -70° C.

EXAMPLE 7 Determination of the DNA Binding Site of mCREBa

The binding specificity of mCREBa for DNA sequences is determined usingBinding Site Selection as described by Pollock and Treisman NucleicAcids Res. 18:6197-6204 (1990)! which permits rapid identification ofthe DNA binding site of a protein from random oligonucleotides. Toperform Binding Site Selection of the mCREBa site, the biotin-mCREBaprotein prepared as described above is incubated with a 26 nucleotiderandom oligomer flanked by specific nucleotides that will anneal tooligonucleotide primers in a PCR reaction. ##STR1## The binding reactionis carried out under conditions of low ionic strength to avoidinterrupting salt-sensitive binding reactions. Binding buffer containingDignam's buffer D Digman, et al., Nucl. Acids Res. 11: 1475-1489 (1983)!with protease inhibitors, 0.1% Nonidet P-40, 1 mg/ml acetylated bovineserum albumin (Promega), is combined with 200 ng Poly (dIdC)-Poly (dIdC)(Pharmacia), 1 pg biotin-mCREBa, and 0.4 ng random oligonucleotide andincubation carried out for thirty minutes on ice to permit protein/DNAcomplex formation. Streptavidin agarose is added to the binding reactionand following incubation overnight at 4° C. with rocking, mCREBa/DNAcomplexes formed are collected by centrifugation. Complexes are washedtwo times with the above binding buffer and DNA eluted from complex byincubation at 45° C. in buffer containing 200 μl 5 mM EDTA, 0.5% SDS,100 mM sodium acetate, 50 mM Tris, pH 8.0. DNA is phenol extracted,mixed with 10 μg of rabbit muscle glycogen (Boehringer Mannheim) ascarrier, and ethanol precipitated. Recovery of DNA is quantitated byCerenkov counting.

The recovered DNA is amplified in a 10 μl PCR reaction including 150 μgeach of primers F and R (SEQ ID NOs: 8 and 9, respectively), 5 μCi ³²P-dCTP, 20 μM dCTP, 50μ each dATP, dGTP, dTTP, 1 mg/ml bovine serumalbumin, 1× PCR buffer (Perkin-Elmer Cetus), 1.5 mM MgCl₂, and 1 unitAmpliTaq (Perkin-Elmer Cetus). ##STR2## Amplification is carried outwith an initial denaturation incubation at 94° C. for four minutesfollowed by 15 cycles of 94° C. for one minute, 62° C. for one minute,and 72° C. for one minutes. The product of this PCR reaction is gelpurified by after electrophoresis on a 8% nondenaturing polyacrylamidegel and used (instead of the random oligonucleotide) for reselection ofDNA binding sequences as described above. This may be repeated 3 or 4times. The DNA final products are digested with BamHI and EcoRI whichflank the defined DNA binding site and cloned into the BamHI and EcoRIsites in the polylinker region of the vector pGL2-promoter (Promega).Plasmid pGL2promoter is a vector that expresses firefly luciferase undercontrol of the SV40 early promoter with a polylinker region upstreamfrom the promoter that permits insertion of potential regulatorysequences. The resulting plasmids are subjected to DNA sequence analysisto determine a consensus nucleotide sequence of the DNA binding site formCREBa.

DNA binding of mCREBa to the defined sequence is confirmed using a gelmobility shift assay. Briefly, purified biotin-mCREBa is incubated for15 minutes at 23° C. with a 0.1 pmol/μl ³² P-labelled oligonucleotidecontaining a DNA binding site identified as described above in buffercontaining 50 mM Tris, pH8, 50 mM NaCl, 50 μM DTT, 1 mM EDTA, 10%glycerol, 5 mM MgCl₂, 5 mM spermidine, 0.05% Nonidet P-40, 3 μg bovineserum albumin, and 1 μg poly(dIdC)-poly(dIdC) (Pharmacia). Afterappropriate incubation, the binding mixture is loaded onto a 4%nondenaturing polyacrylamide gel and mCREBa binding to a radiolabelledDNA sequence determined by a higher molecular weight shift of the DNA ascompared to control DNA in the absence of mCREBa protein.

As further characterization of mCREBa binding to previously identifiedDNA sequences, pGL2 constructs containing the mCREBa DNA binding site iscotransfected into 293T cells with pcDNA3-mCREBa and the ability ofexpressed mCREBa to either activate or repress transcription of thereporter construct is determined. Cells are transfected by the method ofChen and Okyama supra!, harvested after 48 hours and assayed forluciferase activity using a Luciferase Assay Kit (Promega) according tomanufacturer's suggested protocol.

EXAMPLE 8 High Throughput Screen for Modulators of mCREBa DNA BindingActivity

An assay for high throughput screening of small molecule inhibitors(combinatorial libraries, natural product libraries, and/or chemicallibraries) is established based on the defined DNA binding site ofmCREBa determined above. A filter binding assay is designed in which theability of recombinant mCREBa to bind to a ³² P-labeled DNA sequence ismonitored by quantitating radioactivity bound to protein immobilized onnitrocellulose filter. The ³² P-labeled DNA is designed to besufficiently small in order that it will not bind to nitrocellulose inthe absence of previously immobilized the mCREBa protein. Putativemodulators are incubated with the immobilized protein and modulators ofDNA binding activity are identified as those which effect an increase ordecrease in the ability of the DNA to bind to the protein.

An alternative assay is established in which recombinant biotinylatedmCREBa is bound to streptavidin-coated plates (Pierce), incubated withcandidate small molecule modulators and the ³² P-labeled DNA sequenceadded. Modulators are difined as those molecules that increase ordecrease binding of mCREBa to the labeled DNA sequence.

Secondary assays invlove treating cells transfrected withpGL2-promoter/DNA binding site constructs with defined modulatorsfollowed by assay of activity of the product of the reporter gene.

EXAMPLE 9 Identification of Modulators of mCREBa Binding to OtherProteins

Co-pending U.S. patent application Ser. No. 08/721,730 describes indetail a "split hybrid assay" technique to identify molecules whichdisrupt specific protein/protein interaction. The specification of thatapplication is incorporated herein by reference.

In order to isolate cDNAs which encode proteins that interact with CKIδ,the two hybrid assay was performed using a LexA-CKIδ fusion protein asbait. The coding region of CKIδ was subcloned into a BamHI site ofpBTM116 and transformed into a yeast strain designated CKIδ/L40 (MAT ahis3 Δ200 trp1-901 leu2-3 112 ade2 LYS::(1lexAop)₄ HIS3 URA3::(1lexAop)₈-1cZ GAL 4). CKIδ/L40 was subjected to a large scale transformation witha cDNA library made from mouse embryos staged at days 9.5 and 10.5.Approximately 40 million transformants were obtained. Eighty-eightmillion were plated onto selective media lacking leucine, tryptophan andhistidine. The ability of yeast transformants to grow in the absence ofhistidine suggested that there was an interaction between CKIδ and somelibrary protein.

In a second screening, interaction of the two proteins was assayed bythe ability of the interaction to activate transcription ofβ-galactosidase. Colonies that turned blue in the presence of X-gal werestreaked onto media lacking leucine, tryptophan and histidine, grown upin liquid culture and pooled for isolation of total DNA. Isolated DNAwas used to transform E. coli strain 600 which lacks the ability to growon media lacking leucine. Colonies that grew were used for plasmidpreparation and three classes of cDNA were identified. One class wasclosely related to a Drosophila transcription factor dCREBa.

When CKIδ/CREB interaction was examined in the split hybrid assay, cellswere shown to grow on media containing histidine, but in the absence ofhistidine, growth was inhibited. Addition of small amounts oftetracycline to the cell culture restored the cell's ability to grow,suggesting that the interaction between CKIδ and CREBa was very weak.

In order to identify molecules capable of disrupting binding interactionbetween mCREBa and CKIδHU, the split hybrid assay is employed in thepresence of putative inhibitors selected from, for example, chemicallibraries, natural product libraries, and combinatorial libraries.Similarly, inhibitors of interaction between mCREBa and otherinteracting kinases, as well as other proteins, are also identified inthe same techniques. Expression plasmids for other mCREBa-interactingproteins are constructed using techniques similar to those used inconstruction of a CKIδHU expression plasmid. The split hybrid assay isthen performed in the presence of various putative binding inhibitorswherein inhibition is determined by the ability of the host cell to growon media lacking a nutritional requirement.

In the split hybrid assay, mCREBa, or a protein binding domain fragmentthereof, is expressed in a host cell as a fusion protein in combinationwith either a DNA binding or transactivating domain of one or moretranscription factors. Examples of mCREBa binding partners include cdc2see review in Hall and Peters, Adv. Cancer Res. 68:67-108 (1996)!, CKIand CKII isoforms see review in Ahmed, Cell. Mol. Biol. Res. 40:1-11(1994)!, MAP kinase see review in Marshall, Ann. Oncol. 6: Suppl. 1:63-67 (1995)!, and S6 kinase see review in Chou and Blenis, Curr. Opin.Cell. Biol. 7:806-814 (1995)!. 814 A natural binding partner of mCREBais also expressed in the same host cell as a fusion protein incombination with either a DNA binding or transactivating domain of atranscription factor (whichever is not incorporated into the mCREBafusion protein). Expression of the two fusion proteins, and subsequentassociation of the binding partners permits association of the DNAbinding and transactivating domains forming an active transcriptionfactor that leads to expression of a repressor protein. The expressedrepressor protein, in turn, prevents expression of a reporter gene, thussignificantly decreasing survival of the host cell. The thus transformedhost cells can then be contacted with putative inhibitors ofmCREBa/binding partner interaction and actual inhibitors identified asthose which prevent mCREBa binding to its partner protein, thuspreventing expression of the prepressor protein which cannot thereforeblock expression of the reporter gene. An inhibitor therefore indirectlyprovides for a positive signal generated by expression and detection ofthe particular reporter gene product. Sources of potential inhibitorsamenable to this type of assay can be found in chemical compoundlibraries, libraries of natural products isolated from microorganisms,animals, plants, and/or marine organisms, multiparallel syntheticcollections, and/or combinatorial, recombinatorial, peptidomimetic,peptide, polypeptide or protein libraries.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3190 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 304..1866                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCACGAGGGACTTTCTTGGGATGAGCGCTGCCTTTTTGGCTTCCTTTTGGATGCACAGC60                CCGATTTAACCCCTGCACCTTCCGCCCGATCCCAGCAGGCTTGTCCTCCCCGGGGAGTCA120               CAGATTTCCGAGGACAAGGGTCGCGTAGCCTTCGGCAGGGCTCTCCCGAGTTCCTGCTCC180               AGTGCATAAGTTCCACGCGCGCACACGCCAAGTACACGGGGAGAAGCGTCTCACCGGCCC240               GCGGCGGCTCTGCGCGGTCCCCTCCTGCCTCAGCATCCTCGGGCCTGCGCGGCGCCCACC300               GCCATGGAGGTGCTGGAGAGCGGGGAGCAGAGCGTCCTGCAGTGGGAC348                           MetGluValLeuGluSerGlyGluGlnSerValLeuGlnTrpAsp                                 151015                                                                        CGCAAGCTGAGCGAGCTGTCAGAGCCCGGAGAGACTGAGGCCCTCATG396                           ArgLysLeuSerGluLeuSerGluProGlyGluThrGluAlaLeuMet                              202530                                                                        TACCACACGCACTTCTCGGAGCTCCTAGACGAGTTTTCCCAGAACGTC444                           TyrHisThrHisPheSerGluLeuLeuAspGluPheSerGlnAsnVal                              354045                                                                        CTGGGTCAGCTCCTGAGTGACCCTTTCCTCTCAGAGAAGAGCGAGTCA492                           LeuGlyGlnLeuLeuSerAspProPheLeuSerGluLysSerGluSer                              505560                                                                        ATGGAGGTGGAGCCATCTCCAACATCACCAGCGCCTCTCATCCAGGCT540                           MetGluValGluProSerProThrSerProAlaProLeuIleGlnAla                              657075                                                                        GAACACAGCTACTCTCTGAGCGAGGAGCCCCGGACTCAGTCACCATTT588                           GluHisSerTyrSerLeuSerGluGluProArgThrGlnSerProPhe                              80859095                                                                      ACCCATGCGGCTACCAGCGACAGCTTCAATGACGAGGAGGTGGAGAGT636                           ThrHisAlaAlaThrSerAspSerPheAsnAspGluGluValGluSer                              100105110                                                                     GAAAAATGGTACCTGTCTACAGAGTTTCCTTCAGCTACCATCAAGAAA684                           GluLysTrpTyrLeuSerThrGluPheProSerAlaThrIleLysLys                              115120125                                                                     GAGCCAATCACAGAGGAGCAGCCCCCGGGACTTGTCCCTTCTGTCACT732                           GluProIleThrGluGluGlnProProGlyLeuValProSerValThr                              130135140                                                                     CTGACCATCACAGCCATTTCCACTCCTTTTGAAAAAGAAGAGTCCCCT780                           LeuThrIleThrAlaIleSerThrProPheGluLysGluGluSerPro                              145150155                                                                     CTGGATATGAATGCTGGGGGGGACTCCTCATGCCAGACGCTTATTCCT828                           LeuAspMetAsnAlaGlyGlyAspSerSerCysGlnThrLeuIlePro                              160165170175                                                                  AAGATTAAGCTGGAGCCCCACGAAGTGGATCAGTTCTTAAACTTCTCC876                           LysIleLysLeuGluProHisGluValAspGlnPheLeuAsnPheSer                              180185190                                                                     CCGAAAGAAGCCTCCGTGGATCAACTGCACTTACCACCAACACCACCC924                           ProLysGluAlaSerValAspGlnLeuHisLeuProProThrProPro                              195200205                                                                     AGTAGTCACAGCAGTGACTCTGAGGGCAGCTTGAGCCCCAACCCACGC972                           SerSerHisSerSerAspSerGluGlySerLeuSerProAsnProArg                              210215220                                                                     CTGCATCCCTTCAGCCTGTCTCAGGCCCACAGCCCTGTCAGAGCCATG1020                          LeuHisProPheSerLeuSerGlnAlaHisSerProValArgAlaMet                              225230235                                                                     CCCCGGGGCCCCTCTGCCTTGTCCACATCTCCTCTCCTCACAGCTCCA1068                          ProArgGlyProSerAlaLeuSerThrSerProLeuLeuThrAlaPro                              240245250255                                                                  CATAAGCTGCAGGGATCGGGCCCCCTGGTCCTGACAGAAGAGGAGAAG1116                          HisLysLeuGlnGlySerGlyProLeuValLeuThrGluGluGluLys                              260265270                                                                     AGGACCCTGGTTGCCGAGGGCTATCCCATTCCCACCAAGCTGCCTCTG1164                          ArgThrLeuValAlaGluGlyTyrProIleProThrLysLeuProLeu                              275280285                                                                     ACAAAATCTGAGGAGAAGGCCCTGAAGAAAATCCGGAGAAAGATCAAG1212                          ThrLysSerGluGluLysAlaLeuLysLysIleArgArgLysIleLys                              290295300                                                                     AATAAGATTTCTGCCCAAGAAAGCAGGAGAAAGAAGAAAGAATACATG1260                          AsnLysIleSerAlaGlnGluSerArgArgLysLysLysGluTyrMet                              305310315                                                                     GACAGCCTGGAGAAAAAAGTGGAGTCTTGTTCAACTGAGAACTTGGAG1308                          AspSerLeuGluLysLysValGluSerCysSerThrGluAsnLeuGlu                              320325330335                                                                  CTTCGGAAGAAGGTGGAGGTGCTGGAGAACACCAATAGGACTCTCCTT1356                          LeuArgLysLysValGluValLeuGluAsnThrAsnArgThrLeuLeu                              340345350                                                                     CAGCAACTTCAGAAGCTTCAGACTTTGGTGATGGGGAAGGTCTCTCGA1404                          GlnGlnLeuGlnLysLeuGlnThrLeuValMetGlyLysValSerArg                              355360365                                                                     ACCTGCAAGTTAGCTGGCACACAGACTGGCACCTGCCTCATGGTCGTT1452                          ThrCysLysLeuAlaGlyThrGlnThrGlyThrCysLeuMetValVal                              370375380                                                                     GTGCTTTGCTTTGCTGTTGCATTTGGAAGCTTCTTTCAAGGCTATGGG1500                          ValLeuCysPheAlaValAlaPheGlySerPhePheGlnGlyTyrGly                              385390395                                                                     CCTTATCCTTCTGCCACCAAGATGGCTCTGCCCAGCCAGCATCCTCTG1548                          ProTyrProSerAlaThrLysMetAlaLeuProSerGlnHisProLeu                              400405410415                                                                  TCAGAGCCATACACAGCCTCCGTGGTGAGATCCAGGAACCTGCTAATC1596                          SerGluProTyrThrAlaSerValValArgSerArgAsnLeuLeuIle                              420425430                                                                     TATGAGGAACACGCTCCCCTGGAAGAGTCGTCGAGCCCAGCCTCAACC1644                          TyrGluGluHisAlaProLeuGluGluSerSerSerProAlaSerThr                              435440445                                                                     GGGGAGCTGGGGGGCTGGGACAGAGGCTCCTCTCTGCTCAGGGCATCG1692                          GlyGluLeuGlyGlyTrpAspArgGlySerSerLeuLeuArgAlaSer                              450455460                                                                     TCGGGGCTTGAGGCCCTGCCAGAGGTGGATCTTCCCCATTTCCTTATC1740                          SerGlyLeuGluAlaLeuProGluValAspLeuProHisPheLeuIle                              465470475                                                                     TCCAATGAGACGAGCTTGGAGAAGTCAGTACTGTTGGAGCTTCAGCAG1788                          SerAsnGluThrSerLeuGluLysSerValLeuLeuGluLeuGlnGln                              480485490495                                                                  CACCTGGTCAGCAGCAAACTGGAAGGGAACGAAACACTCAAGGTTGTA1836                          HisLeuValSerSerLysLeuGluGlyAsnGluThrLeuLysValVal                              500505510                                                                     GAGCTGGAGAGGAGAGTGAACGCCACCTTCTGAGGAGAGCTCCACCCTCC1886                        GluLeuGluArgArgValAsnAlaThrPhe                                                515520                                                                        TCTTCTCCTAACTCCATCTGATCGTCCTTTCAGTTTCCCCTTCACCACTGGATCTCGAGG1946              AGGAGATGGCTAGTGTTACGGCTCGAGACAGGAGGCCAGCCCAGGGGGTTCTGCTTATGT2006              GTCCCCGTGGCTCTCCACAAAAGGGAGCTAGCACCTCTCCATCCCTTTCTCTTACTGCCA2066              TTGGAAATTATTTTAGGGCTGAGATAGGGGTGGAACGAGCAGGCTTGTTTCCACCAATAG2126              TGCCAAGAAGACACTGCCTGATTCTTCCCCGGGAGGAGTGACTCCTCTGAAGAAGACATG2186              ACTCATGTTCAGTTGAGACCCCAGACTCTAGCCACACACATGCCACAGACATGCCAGGGA2246              GTGGCAAAGCACTGACTCCTGAGCTCCCTTCCTCACTAGGACTCCAGTGTGACCCTGCAC2306              TGAGAGGACCAAAGCGTCATTGCAGTCTTCTCTCCACCCTGTACCCCGGAGTCCTGATTG2366              GATGTCTGCAGAGGCAGATGGGGCTCCCACCATATTTTCAGGCCGCAAGTGCAATTCCTG2426              AAGGCATCAGGCTCTTCTCTCCCAGGCTCTCCTGCCCACTGTGTTGTTTGTAGGACACCC2486              CCACACCCACTCATACACAGCCTGCATCTCCACAGGACAATAGCTCTGTCTCCCTGGCCT2546              CCCCTCCCCATTTGTAAATAGTATTTATTAGCTTGCTCAAGCTCCCAGCTGGCCATAGTG2606              AAAAGATTTCCCCTTTCAACCAGCAAAGTCTTCTGTTGGCCTTTGGAACAGGAGAGTCCC2666              CGGAATCTAGGACCCTAGTCTTTGTACTTGATGCCTTGTTTCCCCCCTTTTCTTCTTTAA2726              AATTGGGGACCTATAACATCATCGCTGTTGCGGAATCCACTTAGGCATGTGTCCCCTGAT2786              GGATGAATACATGGGAATGGTGGATACTGTCTTCTGACTCAGGCTCTAGGCTCCATGGCT2846              TCCTCTCTCTGGTCCTGCCACACAGAAGGAAAGCCCTGTCCAGGATAATGAGCGTTGCTG2906              ACACCCTTGCTAGCTTGTCCTGCCTACCTGCTTACCCCACTCCCTCACCTTCCTCCTTCC2966              CTTCTGCCCTCCATCCACCTGCCTTAACTAATTGGGGCTGGAGTTGGTCATTTTTTGTAC3026              ACCCACAGTGGTACCTTTTACAGTCAGGTTTGGATACTTTGCAGCTCATCCAAAGAGACA3086              TAACTAAACCCTAAACTCTTTTTTTGTTGTTGTTGTTGTTGTTTTTTTTTTTTATGATTA3146              AAAAGTAAAAATTGTAGTTTAAAAAAAAAAAAAAAAAACTCGAG3190                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 521 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGluValLeuGluSerGlyGluGlnSerValLeuGlnTrpAspArg                              151015                                                                        LysLeuSerGluLeuSerGluProGlyGluThrGluAlaLeuMetTyr                              202530                                                                        HisThrHisPheSerGluLeuLeuAspGluPheSerGlnAsnValLeu                              354045                                                                        GlyGlnLeuLeuSerAspProPheLeuSerGluLysSerGluSerMet                              505560                                                                        GluValGluProSerProThrSerProAlaProLeuIleGlnAlaGlu                              65707580                                                                      HisSerTyrSerLeuSerGluGluProArgThrGlnSerProPheThr                              859095                                                                        HisAlaAlaThrSerAspSerPheAsnAspGluGluValGluSerGlu                              100105110                                                                     LysTrpTyrLeuSerThrGluPheProSerAlaThrIleLysLysGlu                              115120125                                                                     ProIleThrGluGluGlnProProGlyLeuValProSerValThrLeu                              130135140                                                                     ThrIleThrAlaIleSerThrProPheGluLysGluGluSerProLeu                              145150155160                                                                  AspMetAsnAlaGlyGlyAspSerSerCysGlnThrLeuIleProLys                              165170175                                                                     IleLysLeuGluProHisGluValAspGlnPheLeuAsnPheSerPro                              180185190                                                                     LysGluAlaSerValAspGlnLeuHisLeuProProThrProProSer                              195200205                                                                     SerHisSerSerAspSerGluGlySerLeuSerProAsnProArgLeu                              210215220                                                                     HisProPheSerLeuSerGlnAlaHisSerProValArgAlaMetPro                              225230235240                                                                  ArgGlyProSerAlaLeuSerThrSerProLeuLeuThrAlaProHis                              245250255                                                                     LysLeuGlnGlySerGlyProLeuValLeuThrGluGluGluLysArg                              260265270                                                                     ThrLeuValAlaGluGlyTyrProIleProThrLysLeuProLeuThr                              275280285                                                                     LysSerGluGluLysAlaLeuLysLysIleArgArgLysIleLysAsn                              290295300                                                                     LysIleSerAlaGlnGluSerArgArgLysLysLysGluTyrMetAsp                              305310315320                                                                  SerLeuGluLysLysValGluSerCysSerThrGluAsnLeuGluLeu                              325330335                                                                     ArgLysLysValGluValLeuGluAsnThrAsnArgThrLeuLeuGln                              340345350                                                                     GlnLeuGlnLysLeuGlnThrLeuValMetGlyLysValSerArgThr                              355360365                                                                     CysLysLeuAlaGlyThrGlnThrGlyThrCysLeuMetValValVal                              370375380                                                                     LeuCysPheAlaValAlaPheGlySerPhePheGlnGlyTyrGlyPro                              385390395400                                                                  TyrProSerAlaThrLysMetAlaLeuProSerGlnHisProLeuSer                              405410415                                                                     GluProTyrThrAlaSerValValArgSerArgAsnLeuLeuIleTyr                              420425430                                                                     GluGluHisAlaProLeuGluGluSerSerSerProAlaSerThrGly                              435440445                                                                     GluLeuGlyGlyTrpAspArgGlySerSerLeuLeuArgAlaSerSer                              450455460                                                                     GlyLeuGluAlaLeuProGluValAspLeuProHisPheLeuIleSer                              465470475480                                                                  AsnGluThrSerLeuGluLysSerValLeuLeuGluLeuGlnGlnHis                              485490495                                                                     LeuValSerSerLysLeuGluGlyAsnGluThrLeuLysValValGlu                              500505510                                                                     LeuGluArgArgValAsnAlaThrPhe                                                   515520                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CGCGGATCCTAATGGAGCTGAGAGTCGGG29                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGCGGATCCGCTCATCGGTGCACGACAGA29                                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGGGATCCTCACAGCTCCACATAAGCTGC29                                               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGAATTCGCTCAAGGAGAGTCCTATTGG28                                                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 154 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CAGGTCAGTTCAGCGGATCCTGTCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN60                NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN120               NNNNNNNNNGAGGCGAATTCAGTGCAACTGCAGC154                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CAGGTCAGTTCAGCGGATCCTGTCG25                                                   (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCTGCAGTTGCACTGAATTCGCCTC25                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GAATCGGGCCGCCGAGATCTCATATGGAGCTGAGAGTC38                                      __________________________________________________________________________

What is claimed is:
 1. A polynucleotide encoding a polypeptidecomprising the mCREBa amino acid sequence set forth in SEQ. ID. NO: 2.2. The polynucleotide according to claim 1 comprising the sequence setforth in SEQ ID NO:
 1. 3. The polynucleotide of claims 1 or 2 which is aDNA molecule.
 4. The DNA of claim 3 which is a cDNA molecule.
 5. The DNAof claim 3 which is a genomic DNA molecule.
 6. The DNA of claim 3 whichis a wholly or partially chemically synthesized DNA molecule.
 7. Aexpression construct comprising the polynucleotide according to claim 1.8. A host cell transformed or transfected with the polynucleotideaccording to claim 1 or
 7. 9. A method for producing an mCREBapolypeptide comprising the steps of:a) growing the host cell accordingto claim 4 under conditions appropriate for expression of the mCREBapolypeptide and b) isolating the mCREBa polypeptide from the host cellof the medium of its growth.